Rare mutations in the beta-globin gene and their clinical phenotypes

INTRODUCTION

Beta-thalassemia is one of the most common inherited genetic disorders and has been extensively studied and researched worldwide. The repertoire of mutations in the beta-globin gene is ever expanding and, presently, there are more than 900 mutations in the beta-globin gene, of which 200 mutations are commonly found worldwide.^[1] Mutations may either be clinically silent or lead to the reduced or absent synthesis of the protein, resulting in phenotypes ranging from severe anemia to clinically asymptomatic individuals.^[1] These clinical phenotypes can be predicted by analyzing blood parameters and characterizing hemoglobin (Hb) constituents by high-performance liquid chromatography (HPLC). However, in routine clinical practice, deviations from the expected clinical phenotype necessitate the requirement for molecular analysis and often lead to the discovery of yet new and interesting gene mutations. In India, five common mutations account for 90% of beta-thalassemia and include IVS 1-5G>C (Human genome variation society [HGVS]: HBB: c.92+5G>C), codon 30G>C (HGVS: HBB: c.92G>C), codon 15G>A (HGVS: HBB: c.48G>A), frameshift 41-42(-TTCT) (HGVS: HBB: c.126_129delCTTT), and frameshift 8-9(+G) (HGVS: HBB: c.27dupG).^[2] In the eastern part of the country, Hb E (codon 26G>A; HGVS: HBB: c.79G>A) accounts for a considerable proportion of the thalassemia burden (~5–10% carrier prevalence in the population data).^[3] Our Institute in West Bengal is a nodal center for thalassemia control and sees a large footfall of patients referred from all across the state. Apart from amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and gap-polymerase chain reaction (gap-PCR), we perform Sanger’s sequencing for comprehensive molecular diagnosis of beta-globin gene mutations. Here, we report four patients with rare beta-globin gene mutations and describe their unique clinical phenotypes.

MATERIALS AND METHODS

This retrospective data analysis was conducted at the Institute of Hematology and Transfusion Medicine, Medical College and Hospital, Kolkata. Over 3 years, from January 2017–October 2020, a total of 160 cases underwent comprehensive molecular diagnostics for thalassemia. Cases with clinical phenotype-HPLC discrepancies (e.g., thalassemia trait requiring transfusions), those referred to us for prenatal diagnosis, or those with un-explained HPLC parameters underwent sequencing when conventional molecular diagnostics (ARMS-PCR and gap-PCR) could not elucidate the genotype. Detailed family history, history of consanguineous marriage, transfusion history, and drug history were elicited from the patients. A thorough physical examination of all the patients was done with regard to hepatosplenomegaly, features of iron overload, achievement of growth and development milestones, and endocrinological evaluation. After taking written informed consent, 3–4 mL of blood was taken in ethylenediaminotetraacetic acid ethylenediaminotetraacetic acid-coated vacutainers. A CBC was done using an SYSMEX XP100 automated hematology analyzer (SYSMEX Corporation, Japan) and a peripheral smear was also examined for cellular morphology. Hb-electrophoresis was performed using a BIORAD Variant II beta-thalassemia short program (BIORAD, USA). Initial screening was done by ARMS PCR for a panel of 22 mutations which included the five most common mutations and 17 other less commonly found mutations in India.^[2,4] Gap-PCR was done for alpha-triplication/quadruplications wherever indicated. Those with a non-contributory ARMS-PCR and/ or gap-PCR report subsequently underwent DNA sequencing of the entire beta-globin gene. Technical details followed were those based on the work done by Thein.^[1] Automated DNA isolation was done using Qiacube (QIAGEN, USA). β-globin gene sequencing analysis was done using an ABI-3500 sequence analyzer (ABI Biosystems, USA).

RESULTS

Out of the 160 cases studied, 124 cases (124/160; 77.5%) were found to be homozygous or compound heterozygous for beta-thalassemia by ARMS-PCR. Heterozygous beta mutations with co-existent alpha-triplications/quadruplications were found in 26 cases (26/160; 16.25%). However, the phenotype of ten cases (10/160; 6.25%) were left un-explained by ARMS-PCR and Gap-PCR; these were then subjected to beta-globin gene sequencing. Sequencing analysis revealed five different types of rare mutations in four patients and is described in detail below. The families of these patients were also screened for the mutations. The spectrum of beta-globin gene mutations identified is detailed in [Table 1].

CASE DETAILS

Case 1

An 18-year-old boy presented to the outpatient department (OPD) with microcytic hypochromic anemia and a history of a total of three units of packed cell transfusion at the age of 5 years (Non-transfusion dependent thalassemia [NTDT] phenotype). He was a single child and had no history of thalassemic individuals in the family. On evaluation, he had pallor (Hb = 8.9 g/dL) and jaundice but no organomegaly, skeletal changes, or growth retardation. Serum ferritin was 362 ng/mL. The patient’s HPLC was homozygous for beta-thalassemia (HbA0 – 0.5%, HbA2 – 3%, and HbF – 100.3% with pre-integration peaks in the chromatogram) [Figure 1a], while ARMS-PCR showed only a heterozygous CD 15 mutation. Not being able to explain his NTDT phenotype, beta-globin gene sequencing was done. Sequencing revealed, in addition to CD 15 mutation, two rare mutations, namely, Hb Monroe (HGVS name: HBB: c.92G>C, AGG>ACG leading to arginine to threonine amino acid change at nucleotide position 70686, codon 30, exon 1) and beta nt-42 (HGVS name: HBB: c.-92C>G; C>G substitution at nucleotide position 70503 in the promoter region, 42 base pair upstream to exon 1) [Table 2 and Figure 2]. Since both these mutations were of β0 type and should have led to a transfusion dependent thalassemia (TDT) phenotype, evaluation for alpha deletion was done and as suspected, a single 3.7kb alpha deletion was found, explaining the NTDT phenotype. XMN1 polymorphism (−158, C>T) was also found in the patient and could explain the high HbF value on HPLC. Beta-globin gene sequencing of the parents revealed the mother to be heterozygous for CD 15 and the father, to harbor both mutations Hb Monroe and beta nt-42 (C>G). The patient is doing well on hydroxyurea supplementation and continues to be on follow-up.

Table 2: Rare mutations and their phenotypes: Details of the patients, their parents, clinical features, blood parameters, and mutations detected.

S. No.	Family details	Age (years)/sex	Transfusion dependence/Clinical features	Hb (g/dL)	Red blood cell count (mill/cumm)	Mean corpuscular volume (Fl)	Mean corpuscular hemoglobin (pg)	Red cell distribution width (%)	HbF (%)	HbA0 (%)	HbA2/HbA2+ HbE*/Variant peak (%)	Common β-globin gene mutations	Rare β-globin gene mutations	Alpha-globin gene triplications/deletions
1.	Patient	18/M	No	8.9	3.64	79.1	24.5	35.2	100.3	0.5	3.0	CD 15 heterozygous	Beta nt-42; Hb Monroe heterozygous	3.7kb alpha deletion
	Mother	34/F	No	10.7	5.42	64	19.7	16	2.1	82.4	5.6	CD 15 heterozygous	-	No
	Father	39/M	No	13.3	5.58	73	23.8	16.1	0.9	82.9	5.6	Not found	Beta nt-42; Hb Monroe heterozygous	No
2.	Patient	2/M	Yes	7.1	3.09	68.3	23	27.4	4.8	78.3	4.3	IVS 1-5 heterozygous	Beta−90 (C>T) heterozygous	No
	Mother	26/F	No	12.3	5.71	69.2	21.5	14.9	1.5	83.5	5.7	Not found	Beta−90(C>T) heterozygous	No
	Father	32/M	No; Mild jaundice+	8.4	5.4	54	16.4	22.1	12	9.9	67.2*	CD 26 heterozygous IVS 1-5(G>C)	-	No
3.	Patient	7/M	Yes	5	2.77	60	18	33.4	40.6	3.3	60.7*	CD 26 heterozygous	Hb Randwick heterozygous	No
	Mother	26/F	No	12.9	5.04	78.8	25.6	14.2	0.8	62.8	26*	CD 26 heterozygous	-	No
	Father	29/M	No	11.9	6.39	61.2	18.6	17.2	0.4	84.6	4.8	Not found	Hb Randwick heterozygous	No
4.	Patient	2/F	No; Cyanosis+	9.2	3.42	91.5	27.8	18	1.8	75.8	3.4/Variant peak$	None	Hb M Saskatoon heterozygous	No
	Mother	26/F	No	14.6	4.46	104.5	33	15	0.2	96.5	3.1	None	-	No
	Father	30/M	No	9.0	4.06	94.8	22.2	17.8	0.6	87.8	2.7	None	-	No

$HbC window: Variant peak – 15% at retention time of 5.08 min. Hb: Hemoglobin, *- HbE

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Figure 1:

Hemoglobin (Hb) HPLC chromatograms of the four cases described in the text. (a) Case 1: Patient with Hb Monroe, Beta nt-42, and CD15 heterozygous mutations. Chromatogram shows beta-thalassemia major picture with 100.3% HbF, 0.5% HbA0, and 3% HbA2. (b) Case 2: Patient having IVS 1-5(G>C) and beta −90(C>T) mutations. Chromatogram shows beta-thalassemia like picture with 5.6% HbA2, 82.9% HbA0, and 0.9% HbF. (c) Case 3: Patient with Hb Randwick and HbE heterozygous mutations. Chromatogram shows 60.7% HbA2+E, 3.3% HbA0 and 40.6% HbF. (d) Patient with Hb-M-Saskatoon heterozygous mutation. Chromatogram shows a variant peak at C-window (15%) with 75.8% HbA0, 3.4% HbA2 and 1.8% HbF. HPLC: High-performance liquid chromatography.

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Figure 2:

Snap-Gene software readout of the sequences showing beta mutation −90 (C>T), Hb Monroe, Hb Randwick, Hb-M-Saskatoon, and beta nt-42 (C>G) mutations. Hb: Hemoglobin.

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DISCUSSION

As sequencing technologies advance and become more accessible and affordable, the repertoire of mutations in thalassemia will continue to expand. In this study, we used Sanger’s sequencing, in addition to ARMS and Gap-PCR, as part of comprehensive genetic diagnosis and could evaluate 154 out of the 160 cases of thalassemia with unexplained clinical features. Six cases (6/160, 3.75%) were left undiagnosed even after sequencing analysis. The spectrum of beta-globin gene mutations found in this study closely match with that reported in the literature from the same geographical region.^[2,5] Alpha-triplication occurring together with beta-thalassemia trait, leading to a TDT phenotype was seen in 26 cases (26/160, 16.25%), which again closely matches with the prevalence in India.^[6,7] We found four cases out of 160 (2.5%) to have rare mutations.

The adolescent in the first case was unique in having a conflicting pattern of homozygous beta-thalassemia on HPLC, but with an NTDT phenotype. Sequencing revealed three mutations, namely, CD 15, Hb Monroe (HBB: c. 92G>C), and beta nt-42 (HBB: −92C>G), along with a single 3.7 kb alpha deletion. Hb Monroe was initially reported in an African-American girl who was transfusion dependent.^[8] This mutation in homozygous state leads to a TDT phenotype and in the heterozygous state, has been reported to interfere with the normal/wild-type allele as it reduces splicing efficiency.^[8-11] Another unique feature of Hb Monroe is cooccurrence with the promoter mutation −92C>G and co-localization on the same allele (downstream and upstream, respectively) of the beta-globin gene. This pair of mutations have been reported in various parts of the world, including India.^[9,12-14] The significance of this co-occurrence is yet unknown. The mutation at the promoter region, beta nt-42 (HBB: −92C>G), has been postulated to be the binding site for transcription factor Egr1, a target for hydroxyurea to increase HbF production.^[12] In our patient, the severity of these mutations was offset by the presence of alpha deletion and XMN1 polymorphism. Both XMN1 polymorphism and the promoter region mutation made it worthwhile to prescribe hydroxyurea to the patient.^[12]

The child in case 2 had a transfusion-dependent phenotype with compound heterozygosity for IVS 1-5(G>C) and −90(C>T) mutations. The former leads to a beta0 state, while the latter is a promoter region mutation (T for C substitution at – 90 position upstream in the CACCC box), leading to a reduction of beta-globin protein synthesis causing a beta+ state.^[15,16] In combination, this child had a beta0/beta+ phenotype, and could explain the thalassemia major phenotype. This mutation and others (−87 [C>G] and −88 [C>T]) in the promoter region are rare and mostly lead to a beta+/beta++ phenotype. Only a few cases of −90(C>T) have been reported previously in India (in West Bengal, Assam, Orissa) and abroad (Portugal).^[17,18] The unique distribution of this mutation may be related to India’s colonial past. In our case, the mother was the carrier of the rare mutation and had a similar hemogram (Hb = 12.3 g%, MCV = 69.2 fl, MCH = 21.5 pg, and RDW = 14.9) and HPLC (HbA0 = 83.3%, HbF = 1.5%, HbA2 = 5.7%) profiles as previously reported in the literature.^[18]

Hb Randwick was detected in case 3 and is found to have a North Italian ethnic background.^[19] This mutation is reported to have normal clinical, HPLC, and hemogram parameters with mild reticulocytosis and may occasionally present with HbH such as inclusions, fragmented, and target cells in peripheral smear.^[19] The mutation is not associated with splenomegaly or jaundice. In our case however, the father was found to carry the mutation and also had a high HbA2 value (4.8%) which is in contrast to that reported in the literature (normal HbA2 values). Another possibility is that the father also harbored another beta-globin mutation which led to the high HbA2 value but could not be detected on genetic analysis. In our patient, this mutation was found in a compound heterozygous state with HbE and the child was transfusion dependent. It is therefore intriguing to see this change in behavior of the mutation when present along with HbE. A corollary may be drawn from the HbE-beta compound heterozygous state which manifests as an NTDT or TDT phenotype, whereas, in isolation, HbE homo/ heterozygous state is clinically benign. To the best of our knowledge, this is the first clinically symptomatic case of Hb Randwick reported in the literature.

The fourth case of Hb-M-Saskatoon was detected in a patient with cyanosis. This slightly unstable variant Hb was first described in 1948 as an autosomal dominant inherited disorder and is associated with decreased oxygen affinity and mild anemia.^[20] This mutation has been previously reported in two families in India.^[21] In Hb HPLC, the presentation can be varied, possibly due to post-translational modification of the Hb moiety. It either elutes solely at the A2 window (recently described sporadic case of Hb-M-Saskatoon from Spain had HbA2+variant of 14%), may coelute at A2 and C windows or may even have unknown peaks near the C-window with retention times ranging from 4.3 to 4.8 min.^[21,22] In our patient, the abnormal Hb eluted at the C-window (15%), while A2 levels were normal. As it is an unstable Hb, cases may manifest with mild hemolysis and consequent reticulocytosis but rarely requires transfusion support.^[22] It is characterized by pseudocyanosis (due to plasma methemoglobin >5 g/dL) which becomes clinically apparent 6–7 months after birth as HbA production reaches its peak. Our patient had similar clinical features when compared to the Spanish report: both were sporadic and presented with anemia and cyanosis. However, the Spanish patient had mild anemia and reticulocytosis without any evidence of hemolysis, while ours did not.^[22] In terms of prognosis, the homozygous state of Hb-M-Saskatoon has not been described in the literature and its behavior is not known, but the heterozygous state is clinically benign, has excellent outcomes, and rarely needs therapy.

CONCLUSION

Rare mutations in thalassemia have myriad presentations. It is even more intriguing to find identical mutations in seemingly disconnected parts of the world, in completely different ethnic groups. Whether these mutations arose simultaneously in different parts of the world or travelled along with the migration of humans from one continent to another still remains unanswered. The study of human genetics in relation to anthropology and history is key to the answer to these questions. In the present day, next-generation sequencing will be an indispensable tool in the hands of the diagnostician to unravel such enigmatic cases. Therefore, we expect yet new and clinically significant mutations to be added to the already vast palette of thalassemia genetics in the years to come.